PROJECT SUMMARY Breast cancer (BrCa) is the second leading cause of cancer related deaths in women and the majority of mortality is due to metastatic BrCa disease. Although our understanding and treatment of advanced breast cancer has improved, the 5-year survival rate remains at a dismal 22%. Two-thirds of all BrCa is positive for estrogen receptor (ESR1, ER), which can be exploited by targeted anti-estrogen therapeutics. Unfortunately, 25% of ER-positive primary disease patients and nearly all ER-positive metastatic BrCa patients will go on to develop treatment refractory disease to these targeted regimens. ER is a prominent component of cancer progression; thus, it is imperative to understand how advance ER-positive patients confer treatment resistance. The Lee-Oesterreich lab recently discovered a novel genomic alternation to ESR1, an in-frame translocation event creating a fusion gene product. A subsequent panel of ESR1 fusions have been discovered and these ESR1 fusions are estimated to be prevalent in at least 1-5% of advanced ER-positive BrCa disease. Importantly, these fusions were identified in patients who developed resistance to hormonal therapy. The functional role of these genomic fusion genes requires further investigation. We will investigate how ESR1 fusions influence cellular proliferation, treatment insensitivity and migration/invasion. Our preliminary data of a fusion stably expressed in a BrCa cell line exhibited enhanced growth and lack of response to hormonal treatment. We will also analyze transcriptional and mechanistic changes invoked by ESR1 fusion expression. Preliminary data of ESR1 fusions transiently transfected into a BrCa cell line exhibited enhanced ER activation compared to parental and wildtype ER expressing cells. Direct mutagenesis of the fusion partner will better elucidate the contribution of ESR1 or the fusion partner to global cellular changes. Lastly, we will study the prevalence of the fusion genes in metastatic refractory disease to determine clinical significance. We will analyze exosomal RNA isolated from patient blood draws to detect presence of known and novel fusions. This proposal will ultimately 1) better analyze the metastatic properties of ESR1 fusions both in the presence and absence of treatment, 2) gain a greater appreciation of ESR1 fusion influence on the transcriptome and cistrome and lastly 3) define fusion prevalence in advance BrCa and detection of novel fusions over the course of a controlled clinical trial utilizing anti-estrogen treatment. Successful completion of these Aims will help inform clinicians and researchers the clinical relevance of ESR1 fusions and if these genomic alternations can serve as biomarkers for identifying treatment resistant breast cancer and rationale for alternative treatment strategies.